RMM Product Matrix - Rapid Microbiology and Rapid Microbiological Methods

RMM Product Matrix - Rapid Microbiology and Rapid Microbiologic...
http://rapidmicromethods.com/files/matrix.php
RMMS FOR MICROBIAL IDENTIFICATION

Company Product Name Scientific Method Applications
Time to Result
Throughput
Sample Size or Type
Sensitivity
Organism Libraries
Workflow
DNA is extracted from cells originating from colonies or from liquid samples. The DNA sample is applied to specific wells of a 96 well-plate, preloaded with sequence primer pairs. After PCR Abbott Laboratories, Ibis Biosciences Division PLEX-ID System Note that this RMM is currently being redesigned and is currently unavailable. amplification, the plate is loaded on the PLEX-ID system where pico-liter Genotyping by Multi Loci Base Composition (MLBC) analysis using PCR and mass spectrometry Identification Bacteria, fungi and viruses. > 750,000 microbial signatures with detection down to the strain level amounts of the PCR products are analyzed in an electrospray TOF mass spectrometer. The base composition for all amplicons present are determined and searched against a database to determine the organisms present. This multilocus approach provides the ability to distinguish between closely related species as well as single nucleotide polymorphisms or mutations. There is no requirement for additional staining, culturing or enrichment steps. Simple mixtures of organisms can also be analyzed. Not for use in diagnostic procedures. DNA is extracted from cells originating from isolated colonies. Amplify target 16S rDNA for bacteria or the D2 region of large-subunit rDNA for fungi using PCR. Perform sequencing of the PCR amplicons, resulting in DNA fragments of various sizes ending in different nucleotides/dyes. A genetic analyzer separates the fragments by size and a laser detects the fluorescence color from each dye, producing a full gene sequence of the target DNA. The resulting sequence is compared with an internal database of known sequences. No Gram staining is required. Sample material is retained on a supported film. The area is examined for microscopic particulates using Raman spectroscopy and a spectral signature is provided for each particulate. The spectral signatures are statistically correlated to a library of known microorganisms. No need for Gram staining. Mixed cultures and be identified and enumerated. Non-destructive for further analysis. Cells from isolated colonies are used to prepare a microbial suspension, which is then added to specific test cards containing a variety of Biolog OmniLog; MicroStation; MicroLog Growth-based; carbohydrate utilization. Identification 2-72 hr 50 per day Cells from colony Not applicable >1226 bacteria and >885 yeast/mold (GEN II cards) carbohydrates and a colorless tetrazolium violet dye. If growth
6-8 hr
250 every 24 hr
40-160 uL of nucleic acid sample
10 copies per gene
Applied Biosystems MicroSEQ
PCR and gene sequencing Identification
4-5 hr
80 per day. Higher with greater capacity capillary analyzers.
>1800 bacteria Cells from colony Not applicable >90 Mycoplasma >1100 yeast and mold
Battelle REBS
Raman spectroscopy Identification and enumeration
Cells from 160 every 8 hr colony, liquid medium, product/raw material, surfaces 1 cell is identified and quantified
Alcaligenes, Pseudomonas, Brevundimonas, Candida, E. coli, Bacillus, Ralstonia, vegetative and spore forms.
3 min
occurs, the dye turns violet in color. The resulting color patterns are >1000 bacteria (GEN III compared with an internal library. card) Gram staining is required when using GEN II test cards for bacteria, yeast and mold. No Gram stain is required for the GEN III bacterial card (ID's both Gram + and - bacteria).
BD Diagnostic Systems Phoenix
Growth-based; biochemical utilization and antibiotic
3 hr
100 per day
Cells from colony
Not applicable
>225 bacteria
Cells from isolated colonies are used to prepare a microbial suspension, which is then added to specific test cards containing substrates (in wells)
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susceptibility Identification
for biochemical utilization. Color or fluorescence changes in each well are compared with an internal library. Gram staining is required to determine the correct test card to use. Cells from isolated colonies are used to prepare a microbial suspension, which is then added to specific test cards containing substrates for 2-18 hr 30-60 per day Cells from colony >285 bacteria Not applicable >48 yeast enzymatic utilization, carbohydrate acidification and other tests. Color or turbidity changes in each well are measured every 15 minutes and results are compared with an internal library. Gram staining is required to determine the correct test card to use. 13 samples per chip in 4 hr. Each 4 hr additional chip (13 samples) every 1 hr. DNA is extracted from cells originating from isolated colonies. Amplify target DNA (noncoding repetitive DNA sequences) using PCR. PCR products are then separated using electrophoresis in a microfluidics chip and the resulting patterns are compared to an internal library. Cells from isolated colonies or liquid medium are added to a stainless steel target plate and allowed to dry. A UV-absorbing matrix is added and the cells are ionized by a laser. The ionized particles are accelerated in an electric field and enter the time of flight (TOF) tube, where protein and peptide molecules are separated according to their mass to charge ratio. The resulting MALDI-TOF mass spectrum is compared with an internal database. Gram staining is not required. Mixed culture ID possible. Cells from isolated colonies or liquid medium are added to a stainless steel target plate and allowed to dry. A UV-absorbing matrix is added and the cells are ionized by a laser. The ionized particles are accelerated in an electric field and enter the time of flight (TOF) tube, where protein and peptide molecules are separated according to their mass to charge ratio. The resulting MALDI-TOF mass spectrum is compared with an internal database. Gram staining is not required. Mixed culture ID possible. Microorganisms are harvested from the cultivation medium, suspended in water and then transferred on a special IR-transparent, reusable sample plate. The measurement is performed after drying of the samples. The dried biofilm is then analyzed by the FT-IR microplate reader. The FT-IR spectrum is then compared with an internal library. MLVA is a PCR based typing method that relies on the inherent variability found in many regions of repetitive DNA called VNTR (Variable Number
bioMrieux Vitek 2 Compact
Growth-based; biochemical and carbohydrate utilization. Identification
bioMrieux DiversiLab
PCR Identification
>168 bacteria Cells from colony Not applicable >1100 strain or sub-species patterns
bioMrieux Vitek MS
MALDI TOF mass spectrometry Identification
2 min
480 every 8 hr
Cells from colony
508 bacteria Not applicable 78 fungi
Bruker Daltonics MALDI Biotyper
MALDI-TOF mass spectrometry Identification
1-2 min
30-60 per hr
Cells from colony or liquid sample
Not applicable
> 2,000 species and > 4,010 strains of bacteria, yeast and mold
Yeast, bacilli, Bruker Daltonics TENSOR 27+HTS-XT Fourier Transform Infrared (FT-IR) Spectrometry Identification Continuous sampling on 96 and 384 plate formats Coryneforms, Micrococci, Staphylococci, Bifido, Clostridia, Pseudomonads, Lactobacilli, Acetobactereaceae
Minutes
Cells from colony
Not applicable
ceeram ceeramTools Genotyping Kits LP
MLVA (Multi Locus VNTR Analysis) Genotyping
2 days
100 samples in 2 days
DNA or culture cell
Not applicable
Tandem Repeat) which represent Legionella sources of polymorphisms. The MLVA pneumophila, assay for L. pneumophila examines Staphylococcus aureus, 12 loci and the assay for S. aureus Pseudomonas and P. aeruginosa examines 16 loci. aeruginosa Thus, each isolate is defined by a 12 or 16-digit numeric code corresponding to the number of repeats at each VNTR. Efficient amplification of the markers is
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performed in a single or 2 multiplex PCR reactions. An ABI sequencer is required to analyze the amplicons. DNA is extracted from cells originating from isolated colonies. The DNA is cut into fragments using a restriction enzyme, which are then separated according to size. The DNA is Ribotyping of DNA fragments Identification >1,440 bacteria 8 hr 32 per day Cells from colony Not applicable >8,500 strain or sub-species patterns immobilized on a nylon membrane, denatured to produce single-stranded DNA, and then hybridized with a DNA probe (derived from an E. coli rRNA operon). An antibody-enzyme conjugate is bound to the probe and a chemiluminescent agent is added, resulting in a banding pattern that is compared with an internal database. Fully automated; no Gram staining required. A microarray based test kit for the detection and identification of mycoplasma species in cell cultures and other biological materials. DNA is PCR and microarray analysis Mycoplasma detection and identification Detection of > 90 species of Mycoplasma and identification of 40 Mycoplasma species extracted and PCR performed using primers specific for conserved and species-specific regions of the 16S-23S rRNA intergenic transcribed spacer (ITS) of Mycoplasma DNA. The fluorescently labeled fragments are then hybridized to the microarray chip. The chip contains probes for both species-specific targets and a universal probe for all Mycoplasma. Fatty acids are extracted from cells originating from isolated colonies. The fatty acids are purified and analyzed using gas chromatography. The resulting GC chromatogram is compared with an internal library. No Gram staining is required. Rack of tubes with suspended cells is inserted into an automated Sample Preparation Module to be processed in parallel. The Module isolates and purifies DNA, specifically digests it, tags DNA with fluorescent probes, and elutes the samples for consecutive measurement with the Detector Module. The fragments of genomic DNA are stretched and fluorescent signatures generated by the probes are measured one-by-one. Each signature is compared with internal database to identify an isolate or elucidate the composition of microbial mixture. The sample material is collected on metal foil. Viability staining and Viable Staining and Imaging LED Raman Spectroscopy Identification and enumeration automated image analysis using dark field illumination detects viable particle quantity, shape, and size for particles ranging from 0.5 m and larger. Raman spectroscopy is then performed on each viable particle, and a spectral signature is provided. The spectral signatures are statistically correlated to a library of known microorganisms. Non-destructive for further analysis. Cells from isolated colonies are incubated in liquid medium for 18 hr, followed by washing, centrifugation 2 hr 350 per day Cells from colony Not applicable Bacteria and resuspension in distilled water. 50 uL of the suspension is transferred onto a special IR-transparent, reusable sample plate and dried at
Dupont Qualicon Riboprinter
Greiner Bio-One CytoInspect
5 hr
100 per day
MIDI Sherlock MIS
Detection of fatty acids. Identification
Standard method (2 hr); Instant FAME method (<15 min)
100 per day
Cells from colony
>1,200 bacteria Not applicable >200 yeast/mold
Pathogenetix, Inc. Genome Sequence Scanning
Fluorescent tagging of single molecules of genomic DNA Identification, analysis of microbial mixtures, epidemiology, contamination source tracing
3.5 - 4.5 hr
50 samples per 24 hr
7 9 0.1% of 10 - 10 cells in 0.1 - 5 mL of microbial target culture or from mixed with other bacteria colony
1,000+ organisms including bacteria, yeast, mold, and Mycoplasma. Library can be customized.
rap.ID Bio Particle Explorer BPE
3-10 min
> 150 samples per 8 hr
Cells from colony, liquid medium, product/raw material, surfaces
1 cell is identified and quantified
> 30 bacteria; can add new entries
Thermo Scientific Nicolet
Fourier Transform Infrared (FT-IR) Spectrometry Identification
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40-45C under vacuum to create a biofilm. The biofilm is then analyzed and biomolecules such as proteins, lipids, carbohydrates and DNA/RNA are identified and quantified. The FT-IR spectrum is then compared with an internal library.
RMMS FOR QUALITATIVE ANALYSIS

Company Product Method Applications
Time to Result
Throughput
Sample Size or Type
Sensitivity
Organisms Detected
Workflow
The test sample is added to unique vials that contain a selective medium and a dye. Changes in color or fluorescence, expressed as light Total aerobic count, intensity units, are detected by the yeast & mold, coliforms, optical sensor and represent growing E. coli, lactic acid microorganisms. The total aerobic bacteria, count and total yeast/mold vials detect Enterobacteriaceae, microbial growth by monitoring the Salmonella, generation of CO , and can be used 2 Pseudomonas, to screen for an estimation of Staphylococcus organisms in a test sample that are above or below a certain quantitative specification. Liquid and diluted solid samples can be assayed. The sample is added to a centrifuge tube and Mycoplasma is concentrated in a pellet. The Mycoplasma is enzymatically lysed and purified using magnetic beads and washing steps. Real time PCR is performed and the Mycoplasma target sequence is detected via use of amplification plots and melt curve analysis. If a positive result fir Mycoplasma is obtained, the purified DNA can be used in the MicroSEQ for subsequent identification. The sample is transferred into a disposable vessel containing a general or selective medium. The vessel is then placed into the portable respirometer and incubated to encourage microbial growth. Detection of metabolic activity is determined by pressure transients relating to gaseous exchanges within the closed culture vessel as a result of microbial Aerobes, facultative anaerobes, anaerobes, and microaerophilic bacteria; yeast respiration. Continuous data collected is analyzed in real time, and detection algorithms process the pressure transients to alert when significant changes have taken place. The instrument measures both positive and negative pressure meaning that monitoring can be performed on a range of microbial processes reacting to differing conditions within the culture chamber. The system is applicable for pharmaceuticals, raw materials, food and beverage, veterinary, household and hygiene products. Samples are added directly to bottles of liquid culture media and incubated
2
Growth-based; CO BioLumix BioLumix System detection and selective growth
32 tests at a single temperature per instrument (32 instruments can be used with one computer) 1 cell after enrichment
Detection of specific organisms; detection of microbial growth
8-48 hr
0.1-1.0 mL
Applied Biosystems MycoSEQ
PCR Mycoplasma detection
< 5 hr
100 l to 10 ml of cell culture sample
< 10 CFU or copy equivalent/ml
Detection of > 90 Mycoplasma species
Respirometry, pressure sensing Bactest Speedy Breedy Detection of microbial growth; sterility testing; presence of specific organisms 1 CFU after enrichment
4-20 hr
2-4 per day
Up to 50 mL
Growth-based; CO BD Diagnostic Systems BACTEC detection Detection of microbial growth; sterility testing
240-1200 per 8-48 hr incubation period 1-10 mL or gm
in the system. During microbial growth, CO in the closed container 1 CFU after enrichment
2
Bacteria, yeast, mold
accumulates and is detected by a fluorometric sensor. The system automatically monitors the sensor every 10 minutes, and the generation of CO indicates the presence of
2
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growing microorganisms. Samples are added to media in a specialized holder that also contain Growth-based; measurement of electrical impedance Detection of microbial growth 64-512 samples per incubation period two electrodes at the bottom of each sample test well. The holder is placed in an incubator and continuously monitored. Growth is detected by monitoring the movement of ions between electrodes (conductance), or the storage of charge at the electrode surface (capacitance). Samples are added directly to bottles of liquid culture media and incubated in the system (one of two temperatures). During microbial growth, CO in the closed container
2 2
bioMrieux Bactometer
6-48 hr
1 mL
1 CFU after enrichment
Growth-based; CO bioMrieux BacT/ALERT 3D Dual-T detection Detection of microbial growth; sterility testing
accumulates and diffuses into a 24-96 hr 480-1440 per incubation period 1-10 mL or gm 1 CFU after enrichment Bacteria, yeast, mold colorimetric sensor at the base of the bottle. Hydrogen ions interact with the sensor resulting in a decrease in pH, causing the sensor to change to a yellow color. The system automatically monitors the sensor every 10 minutes, and the generation of CO indicates
2
the presence of growing microorganisms. Bacterial colonies growing on agar plates are suspended in buffer, and the suspension is placed in a reaction tube. Primers, fluoresence resonance energy transfer (FRET) probes and Taq polymerase are added to the reaction tube. PCR amplification and detection is carried out in a Roche Diagnostics LightCycler 2.0 Carousel-Based System. The amplification cycles are monitorined via fluorescence, and melting curves are used to determine what target sequences/organisms are present. The test sample is filtered/concentrated through a 0.45um micro sieve. Microorganisms are retained on the membrane and subsequently labeled with a viability stain and/or a species-specific stain. Available are total viable count, total live-dead ratio, total species-specific count and total species-specific viable count. After staining a scanning period using MuScan digital fluorescent Filterable samples; 1 uL to 1L 1 - 10 cells
5
BIOTECON Hygiene Screening System
PCR Bacterial detection
90 min
160 per day
Cells from colony
Corynebacterium, Staphylococcus, Macrococcus, Micrococcus, Kocuria, and Kytococcus
Viability staining and solid phase cytometry; fluorescence microscopy Bioburden of raw material and in-process samples, finished product, EM, water, sterility testing
CCM MuScan Innosieve Diagnostics Test Kits
10-65 minutes
100 per 8 hours
Bacteria, yeast, fungal spores
microscopy at specific excitation and emission wavelengths is performed. The image processing software analyzes fluorescent objects on size, shape and fluorescent signals of all microbes. The assay can be user-customized with different types of fluorescent stains, labeled antibodies, DNA-probes, etc., depending on the organism(s) to be detected. Examples of test kits for specific organisms include Legionella, Salmonella, Listeria, Chronobacter and E. coli. The method is non-destructive such that detected microbes can be subsequently cultured.
ceeram ceeramTools RT-PCR Real Time Detection Kits Real time RT-PCR Viral detection 5 hrs Depends on thermocycler used
2 gr digestive tissue, 25 gr fruit or vegetable, 1 L water
Norovirus GI, GII; Hepatitis A, E; 5 genome copies Enterovirus; Adenovirus; Sapovirus; Aichivirus; Rotavirus; Circovirus; Brachyspira; Giardia; Cryptosporidium
Elution, concentration, extraction, purification, amplification, quantification and interpretation. Any matrix (food, environmental, health) may be assayed, including shellfish, vegetable, herbs and spices, water, sludge and surfaces. The detection kits can be used with most PCR instrumentation.
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Celsis International Ltd. Celsis Advance System using RapiScreen
ATP bioluminescence Bioburden of water, raw materials, in-process samples, Microbial Limits <1-48 hr
120 assays per hour
1 CFU in User selected pre-enriched sample Bacteria, yeast, mold
Sample in broth is incubated according to assay type (e.g., MLT, sterility, aerobic/anaerobic, product neutralization). Aliquot of enrichment is placed into cuvette and into instrument. Luciferin/luciferase enzyme reagent catalyzes the conversion of microbial adenosine triphosphate (ATP) into ADP and light. The system provides full walkaway automation. If sufficient cells (and ATP) are present, direct assay can be performed within minutes without broth enrichment. Sample in broth is incubated according to assay type (e.g., MLT, sterility, aerobic/anaerobic, product neutralization). Aliquot of enrichment is placed into cuvette and into instrument. The presence of microbial adenylate kinase catalyzes the conversion of an ADP-containing Bacteria, yeast, mold reagent into ATP at significantly higher levels than microbial ATP alone. Amplified-ATP is then detected by a luciferin/luciferase-based bioluminescence assay resulting in a faster time-to-result and higher signal:noise ratio than traditional ATP bioluminescence. The system provides full walkaway automation.
EnzymeCelsis International Ltd. Celsis Advance System using AMPiScreen amplification combined with ATP bioluminescence Sterility testing, bioburden of water, raw materials, in-process samples, Microbial Limits 30 min (bioburden) 18-24 hr (MLT) 2-6 days (Sterility) 120 assays per hour 1 CFU in User selected pre-enriched sample
Celsis International Ltd. Celsis Rapid Detection System using ReACT
rRNA-based molecular assay with chemiluminescent signal generation Used in conjunction with Celsis RapiScreen or AKuScreen assay to classify type of contamination detected by primary assay
2 hr after completion of primary assay
Based on primary assay
1 CFU in pre-enriched primary sample
Sample of broth enrichment from primary assay is processed using basic lab techniques to yield stable molecular material. Specific probe sets hybridize to any target sequences Gram Negatives, Staph present forming traditional sandwich aureus, Yeast and Mold assay that allows for immobilization (in development), and tagging with chemiluminescent custom probes beacon. Chemiluminescent substrate (ReACTion) is added and in the presence of bound target a light signal is generated that is read by Celsis Advance instrument and reported in relative light units (RLUs). The PTSTM uses LAL kinetic chromogenic methodology that measures a color intensity that is directly related to the endotoxin concentration. in a sample. Each cartridge contains precise amounts of LAL reagent, chromogenic substrate and control standard endotoxin (CSE). Pipette 25 L of a sample into each of the four sample reservoirs of the disposable cartridge. The portable, handheld reader draws and mixes the sample with the LAL reagent in two channels (the Sample Channels) and with the LAL reagent and positive product control in the other two channels (the Spike Channels). The sample is incubated and then combined with the chromogenic substrate. After mixing, the optical density of the wells is measured and kinetically analyzed against an internally-archived standard curve. The MCS system is comprised of five individual spectrophotometers built into a unit with a single USB connector that links to a desktop
Charles River Laboratories EndoSafe-PTS
LAL assay Detection of endotoxin
15 min
4 per hr
25 L
1 - .01 EU/mL range, 5 - .05 EU/mL range, 10 - 0.1 EU/mL range
Endotoxin
Charles River Laboratories EndoSafe-MCS
LAL assay Detection of endotoxin
15 min
20 per hr
25 L
Endotoxin
computer. The MCS uses LAL kinetic chromogenic methodology that measures color intensity directly related to the endotoxin concentration in a sample. Disposable cartridges
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used to run an assay contain precise amounts of LAL reagent, chromogenic substrate and control standard endotoxin (CSE). Pipette 25 L of a sample into each of the four sample reservoirs of a cartridge. The reader draws and mixes the sample with the reagents. After mixing, the optical density of the wells is measured and analyzed against an internallyarchived standard curve. Using a loop, place an isolated colony into 2-3 mLs of saline or LRW (concentration should be 0.5 LAL assay Rapid Gram staining 4 every 3-7 min McFarland equivalence turbidity standard units). Add 25 l each of 4 samples/organisms to 4 channels in the disposable cartridge. Results for Gram +, Gram , or yeast/mold are displayed on the screen of a portable, handheld spectrophotometer. Charles River Laboratories EndoSafe PTS Glucan Assay Portable, handheld spectrophotometer that utilizes disposable cartridges. 30 min 2 per hr 25 L 10-1,000 pg/mL Glucans from yeast and Rapid, in-process test designed for mold investigational purposes to detect (1,3)--D glucans from the cell walls of most yeasts and molds. Samples are enriched in media to provide enough DNA for analysis and to eliminate false positives from dead cells. The enriched samples are heated in a lysis solution to release Salmonella, Listeria DNA. PCR tablets, which contain all monocytogenes, the reagents necessary for PCR plus Camplyobacter fluorescent dye, are hydrated with jejuni/coli, E. coli lysed sample and processed in the O157:H7, Enterobacter cycler/detector. PCR amplifies a DNA sakazakii, fragment that is specific to a target Staphylococcus aureus, organism. The amplified DNA yeast and mold, Vibrio generates a fluorescent signal, and results are displayed as positive or negative results. The system uses SYBR Green, Taqman and Scorpion probes, facilitating the detection of multiple species in a single sample. A microarray based test kit for the detection and identification of mycoplasma species in cell cultures and other biological materials. DNA is extracted and PCR performed using primers specific for conserved and species-specific regions of the 16S-23S rRNA intergenic transcribed spacer (ITS) of Mycoplasma DNA. The fluorescently labeled fragments are then hybridized to the microarray chip. The chip contains probes for both species-specific targets and a universal probe for all Mycoplasma. The system utilizes a highly-sensitive ATP measuring instrument (100 to 200 times higher sensitivity than conventional ATP methods) for ATP bioluminescence Detection of microorganisms detecting airborne bacteria. A unique spore germination induction processing method is used to detect spores. A 1 cubic meter of air is collected within 10 minutes and particles are deposited onto a collection carrier. ATP from dead bacteria are eliminated while ATP from viable bacteria are extracted. An optical detection system measures light intensity within 90 minutes. Hyglos ELISA 3.5 hrs 96 samples every 3 hrs 100 l 0.05-500 EU/mL Endotoxin Uses a microplate that is pre-coated with a phage-derived receptor protein
Charles River Laboratories EndoSafe PTS Gram ID 3-7 min 25 L Gram + and - bacteria
LAL assay Detection of glucan
Dupont Qualicon BAX System Q7
PCR Detection of microorganisms
1.5-2.5 hr; 48 hr for yeast/mold
96 per 1.5-2.5 hr
10-50 L
Greiner Bio-One CytoInspect
PCR and microarray analysis Mycoplasma detection and identification
5 hr
100 per day
Detection of > 90 species of Mycoplasma and identification of 40 Mycoplasma species
Hitachi BioMAYTECTOR
90 min
5-6 samples per 8 hr
Volumetric air sample (e.g., 1 cubic meter)
1 cell (1 attomole ATP)
Bacteria, bacterial spores
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which has a high affinity and specificity for the conserved core region of LPS (endotoxin). When the sample matrix is added to the microplate, the LPS is bound to the EndoLISA Detection of endotoxin phage protein. Any sample matrix with potentially interfering components is then removed by a washing step. The subsequent detection by recombinant Factor C and a fluorescence substrate is left unaffected by inhibitors, facilitating a reliable quantification of endotoxin in the sample. Campylobacter jejuni, Clostridium perfringens, Taqman-based Q-PCR detection kits. Cronobacter sakazakii, When necessary, use pre-enrichment E. coli, Listeria innocua, of the sample in an appropriate Legionella (selective) medium (1:10 w/v: 25 g pneumophila, sample + 225 ml medium) for 20- 24 Legionella spp., Listeria hours at appropriate temperature (the monocytogenes, actual method is provided for each Salmonella sp., test kit). This is followed by filtration of Staphylococcus aureus, 100 ml with an appropriate membrane Vibrio alginolyticus, system (e.g., 0.45 m, cellulose Vibrio cholerae, V. nitrate filter). The detection kits can be cholerae tox, Vibrio used with most PCR instrumentation. parahaemolyticus, Vibrio vulnificus, MRSA The fully automated instrument functions as a real-time, continuous flow water-monitoring system. As the slip stream passes through the flow cell, it also passes through a laser beam. When particles 0.4 microns to 10 microns in size are present, a specific multi-angle light-scatter pattern will be captured by the units photodetector. This pattern is then analyzed and compared to the Bio-Optical Signatures database using proprietary algorithms. From this analysis, relative concentration is calculated and detected particles are classified as a bacteria, spore, protozoan or unknown. The system does not provide viability data as it cannot differentiate between live and dead microorganisms. Viable Mycoplasma are lysed and the organism's enzymes react with the MycoAlert Substrate catalyzing the conversion of ADP to ATP. By measuring the level of ATP in a ATP bioluminescence Mycoplasma detection sample both before and after the addition of the MycoAlert Substrate a ratio can be obtained which is indicative of the presence or absence of Mycoplasma. If these enzymes are not present, the second reading shows no increase over the first, while reaction of mycoplasmal enzymes with their specific substrates in the MycoAlert Substrate, leads to elevated ATP levels. Cells from an isolated colony are suspended in filtered water in a sample vial and placed into the instrument. 35 photo detectors in five concentric arcs that surround the 10 min 48 per 8 hr Cells from colony 10-50 cells E. coli, Cryptosporidium, Giardia sample vial collect Mie scattering light intensities that are generated when a cell intersects a red laser beam. The shape, size and internal/external structures of microorganisms will provide a unique scattering signature, which are compared with an internal database.
< 1 cell / 25 g Innosieve Diagnostics Real-Time Q-PCR Detection Kits (after pre-enrichment) Q-PCR Bacterial detection 3-5 hrs (15-25 cycles) Depends on thermocycler used 25 g < 10 cells / g or 10 non-degraded genomes (no enrichment)
JMAR BioSentry
Light scattering Detection of water pathogens
2 minutes
Continuous monitoring
Process water
600 CFU/mL
Cryptosporidium, Giardia, E. coli, Salmonella, Shigella, Pseudomonas, Legionella, other rod-shaped bacteria
Lonza MycoAlert
20 min
24 per 8 hr
100 L of culture supernatant
< 50 CFU/mL
> 90 species of Mycoplasma
Micro Identification Technologies
Light scattering Detection of microorganisms
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Introduce sample into the centrifugation assay device to separate eukaryotic cells from Mycoplasma. Lysis reagent and primers for the target ribosomal RNA sequence is added. Magnetic particles Merck Millipore MilliPROBE PCR Mycoplasma detection < 1-10 CFU/mL 4 hr 24 per day Up to 20 mL of filterable sample 10,000 copies of Mycoplasma rRNA > 90 species of Mycoplasma capture and purify the RNA target. The purified RNA is added to a 96-well plate, in addition to amplification mix and enzyme reagent. Real-Time Transcription Mediated Amplification (TMA) is performed, and the fluorescence signal from a molecular torch probe increases as amplification occurs. If the Mycoplasma rRNA target is amplified, a positive result is reported. ATP bioluminescence Sterility testing, bioburden (product and surface), non-sterile product release, biological indicator, preservative effectiveness, sanitization monitoring 1 min following 24-48 hr enrichment and membrane filtration steps 100 per day Samples (filterable or non-filterable) are enriched in growth media. Incubated media is then filtered. Microorganisms collected on the membrane are lysed with extractant. 1 CFU after an enrichment of 24 to 48 hr Bacteria (aerobic and anaerobic), yeast and mold Luciferin and luciferase enzyme is added and light is detected with the Pallchek Luminometer. A photomultiplier tube amplifies the photons and results are reported as Relative Light Units (RLU).Additional identification is possible using the residual broth left in the Microfunnel. Samples are filtered and microorganisms collected on the membrane are lysed by a combination of reagents, sonication and heating. The DNA and Master Mix (polymerase and deoxynucleotides) are added to the GeneDisc plate, pre-loaded with E. coli, Salmonella, the primers and probes. The plate is Pseudomonas transferred into the GeneDisc Cycler, aeruginosa, and it rotates through four Staphylococcus aureus, temperature zones during the PCR Candida albicans, amplification process. When the target Aspergillus brasiliensis, DNA sequence (from the STEC and non-STEC, microorganism of interest) is E. coli 0157, Listeria, amplified, a fluorescent signal from Legionella, the probe will increase and is Enterococcus, measured in real time. Current plate Cyanobacteria comes pre-loaded with 6 probes for compendial specified microorganisms. The GeneDisc Cycler can measure fluorescent signal from each probe simultaneously in real time resulting in detection and positive identification of microorganisms in a single PCR run of less than 1 hour. A 1 mL of sample of cell culture with a Roche MycoTool PCR Mycoplasma detection 1 mL of cell < 5 hr culture containing 5 10 cells/mL
6
Pall Corporation Pallchek Rapid Microbiology System
1-300 mL. Protocol adapted for solids and non-filterable samples.
Pall Corporation GeneDisc Rapid Microbiology System
qPCR Screening of pathogens (e.g. Specified Microorganisms, USP <62>)
3 to 8 hours including sample filtration, nucleic acid prep, and PCR
96 per PCR run
1 CFU after enrichment for 6 to 24 hours. >100 CFU without enrichment.
< 1 CFU/mL
> 90 species of Mycoplasma
concentration of 5 10 cells/mL is treated with lysis buffer. Mycoplasma DNA is then purified and the target DNA is amplified via PCR. The resulting amplicons are evaluated using gel electrophoresis. Samples are inoculated directly into a vial which contains ready to use media and a detection system. The optical assay measures microbial growth by monitoring pH or CO that
2
Soleris Neogen
Growth-based, media based detection via CO or

2
Total aerobic count, sterility, yeast & mold, Up to 128 per unit; up to 4 units per computer 1 CFU after enrichment coliforms, E. coli, lactic acid bacteria, Enterobacteriaceae, Pseudomonas, Staphylococcus, aciduric organisms 20 min for quantitative results Up to 8 hr for 96-160 per 8 hrs 75-225 uL 1 CFU after enrichment TB, Chlamydia and Staphylococcus for clinical samples. Salmonella, E. Coli O157, non-O157
pH Quality, spoilage and sterility microbial testing Flow cytometry
3 to 48 hours
0.1-5.0 mL
generate a color change as microorganisms proliferate. Samples and reagent are added to a vial and mixed (5-10 min). The RAPID-B system analyzes the sample using flow cytometry, with results available within 3-5 minutes. Light
Vivione Biosciences, LLC RAPID-B
Incoming inspection, sterility confirmation, bioburden, drug
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scatter resolution of around 130nm shows separation of bacteria populations via size and refractive index alone. The system utilizes a multi-parametric detection approach: a STECs, Staphylococcus, and Vibrio for food samples phenotypic library to identify the bacteria, an immunoprobe to specifically tag the target organism and a DNA dye to ascertain the live or dead cells of the target organism. The workflow is also based on the limit of detection (LOD) required and the target of interest (may include enrichment, filtration, centrifugation and dilution).
development, process control
qualitative results
RMMS FOR QUANTITATIVE ENUMERATION

Company Product Method Applications
Time to Result
Throughput
Sample Size or Type
Sensitivity
Organisms Detected
Workflow
Growth based & staining free; High-Magnification Imaging Bioburden of raw material, in-process samples & finished product, water analysis
Samples are prepared according to the compendial method (membrane filtration or direct inoculation on solid medium). The system incubates the plates (from 20 to 55C) and Filtrable 24-48 hr 240 per 48 hr samples, solid medium 1 CFU after growth Bacteria, yeast, mold, spores automatically acquires high magnified images (X50) of micro-colonies. The image processing software analyzes information on size and shape and provides an enumeration for each plate every 30 minutes. The system is non-destructive, allowing for follow up analysis. Viable cells in a liquid sample are labeled with a non-fluorescent substrate. Within the cytoplasm of metabolically active cells, the substrate is enzymatically cleaved (by esterase) to release a fluorochrome. Cells with intact membranes will retain the fluorescent label. The labeled organisms pass through a argon ion laser in the flow cell. Two fluorescence detectors provide an enumeration in cells per mL. The test sample is filtered through a polyester membrane. Microorganisms retained on the filter are labeled with a non-fluorescent substrate. Within the cytoplasm of metabolically active cells, the substrate is enzymatically cleaved (by esterase) to release a fluorochrome. Cells with intact membranes will retain the fluorescent label. An argon laser scans the surface of the membrane within 3 minutes, and viable cells are detected. Auto-fluorescent particles, membrane fluorescence and background noise are rejected and a total viable count is reported. Viable cells may be subsequently observed using a phase-contrast microscope and an automated stage. Sample material is retained on a supported film. The area is examined for microscopic particulates using Raman spectroscopy and a spectral signature is provided for each particulate. The spectral signatures are statistically correlated to a library of known microorganisms. No need for Gram staining. Mixed cultures and be identified and enumerated.
Advencis Lynx Technology
AES Chemunex D-Count and BactiFlow
Viability staining and flow cytometry Bioburden of raw material and in-process samples, finished product, EM, water
30 min
150 per day (BactiFlow) 300 per day (D-Count)
Liquids, usually less than 1 mL
10-50 cells
Viability staining and solid phase cytometry Bioburden of raw material and in-process samples, finished product, EM, water, sterility testing
AES Chemunex ScanRDI
1.5-3 hr
30 per day
Filterable samples
1 cell
Bacteria, yeast, mold, spores
Raman Battelle REBS spectroscopy Identification and enumeration 3 min 160 every 8 hr
Cells from colony, liquid medium, product/raw material, surfaces
Alcaligenes, 1 cell is identified and quantified Pseudomonas, Brevundimonas, Candida, E. coli, Bacillus, Ralstonia, vegetative and spore forms.
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Non-destructive for further analysis. For a quantitative test, the sample is first diluted in buffer. For a qualitative test (presence/absence testing), the sample is diluted and inoculated into a proprietary enrichment media, which is then incubated for 24-48 hours. Three mL of the diluted sample is then 30 to 10 quantitative; Up to 3 mL 20 samples per hr for qualitative test 1 cell qualitative with enrichment Bacteria, yeast, mold
6
Viability staining and flow cytometry Fermentation growth monitoring, bioburden screening of raw materials, in-process samples, finished product, water analysis, probiotics and stock culture enumeration
4 min quantitative; 24 hr for enrichment based qualitative test (presence / absence)
12-15 per hr for quantitative test;
BD Diagnostics FACSMicroCount
loaded in the instrument for either test. The automated system tags nucleic acid of all microorganisms with fluorescent stain. Sample is then loaded into flow cytometer chamber where a 639 nm red laser hits tagged microorganisms to produce fluorescence and side scatter signals. Signals are captured for each microorganism to reflect one count on intensity plot. Total microbial count is available in both tabular and graphical formats. Air is drawn into the instrument. Particles that pass through a 405 nm diode laser are sized (0.5 to 10 microns) and enumerated using a Mie scattering particle counter. At the same time, particles that contain biological targets, such as NADH, riboflavin, and dipicolinic acid, will auto-fluoresce as they pass through the laser, and a separate fluorescence detector will record these as viable microorganisms. The system; therefore, provides simultaneous viable and total particulate data per cubic volume of air. Results are obtained in real-time, and there are no consumables, reagents or media. The test sample is filtered/concentrated through a 0.45um micro sieve. Microorganisms are retained on the membrane and subsequently labeled with a viability stain and/or a species-specific stain. Available are total viable count, total live-dead ratio, total species-specific count and total species-specific viable count. After staining a scanning period using MuScan digital fluorescent microscopy at specific excitation and emission wavelengths is performed. The image processing software analyzes fluorescent objects on size, shape and fluorescent signals of all microbes. The assay can be user-customized with different types of fluorescent stains, labeled antibodies, DNA-probes, etc., depending on the organism(s) to be detected. Examples of test kits for specific organisms include Legionella, Salmonella, Listeria, Chronobacter and E. coli. The method is non-destructive such that detected microbes can be subsequently cultured. Microorganisms are captured from
BioVigilant IMD-A
Light scattering Active air monitoring
Instantaneous
Continuous or episodic monitoring
IMD-A 350 (28.3 L/min) 1 cell IMD-A 300 (1.15 L/min)
CCM MuScan Innosieve Diagnostics Test Kits
Viability staining and solid phase cytometry; fluorescence microscopy Bioburden of raw material and in-process samples, finished product, EM, water, sterility testing 100 per 8 hours Filterable samples; 1 uL to 1L 1 - 10 cells
5
10-65 minutes
Bacteria, yeast, fungal spores
Lonza MicroCompass II Note that this RMM is currently being redesigned and is currently unavailable.
RT-PCR Estimation of cell count
filterable samples via spin filtration, and are then chemically lysed to release nucleic acid. Alternate procedures are used for non-filterable 4-5 hr 72 per 8 hr ~ 1 mL 100 cells Bacteria, yeast, mold samples. Nucleic acid extraction and purification is fully automated using magnetic beads and washing steps. Purified DNA is amplified using PCR and ribosomal RNA is amplified using reverse transcriptase (RT) PCR. PCR is monitored in real-time (via a MGB
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Eclipse probe), and the number of cycles where the fluorescence rate increases above a threshold value is used to estimate the number of viable cells in the original sample. Appropriate studies are required to correlate the number of PCR cycles with cell concentration. Utilizes a membrane filter to capture individual cells. Filter the sample and Growth-based; ATP bioluminescence Bioburden of raw material and in-process samples, finished product, EM, water, sterility place the membrane on an appropriate agar medium to allow the growth of micro-colonies. Microcolonies are then treated with an ATP-releasing reagent, followed by the addition of luciferin and luciferase. Photons of light from each microcolony are detected by a luminometer, and a cell count is reported. May be able to continue incubation to form larger colonies for subsequent microbial identification. Filter the sample, and incubate the membrane an an agar cassette to form micro-colonies. Add non-fluorescent substrate and Growth-based; viability staining and cellular fluorescence Bioburden of raw material and in-process samples, finished product, EM, water, sterility incubate an additional 30 min. Within the cell, the substrate is enzymatically cleaved, releasing a free fluorochrome into the microorganism cytoplasm. As fluorochrome accumulates inside the cells, the signal is naturally amplified. The membrane is placed in a reader and exposed to the excitation wavelength of the fluorochrome. Fluorescent micro-colonies are then automatically enumerated. Non-destructive; can continue to incubate media to obtain colonies for microbial identification. Samples are filtered and microorganisms collected on the membrane are lysed by a combination of reagents, sonication and heating. DNA is purified and concentrated by further filtration. The DNA and Master Mix (polymerase and deoxynucleotides) are added to the GeneDisc plate, preloaded with the primers and probes. The plate is transferred into the GeneDisc Cycler, Pseudomonas and it rotates through four aeruginosa, temperature zones during the qPCR Staphylococcus aureus, amplification process. With each Candida albicans, amplification cycle a fluorescent signal Aspergillus brasiliensis, is generated from the probe. The point STEC, E. coli 0157, at which the signal reaches above the background is called Cycle Threshold Listeria, Legionella, (Ct) value. The higher the DNA copy Enterococcus, Cyanobacteria (e.g., higher microorganisms), the less amplification cycles will be required to reach the Ct value (i.e., faster detection). Built-in software calculates the number of genomic copies (e.g., amount of DNA, which is directly related to the number of microbial cells/CFUs) present in the initial sample. Identification is possible by DNA sequencing of the remaining DNA sample. Particle Measuring Systems BioLaz Real-Time Microbial Monitor Light scattering Active air monitoring Air is drawn into the instrument sensing area via a stainless steel sample probe. As the air passes through the system it is illuminated by a laser. Biological particles that contain NADH or riboflavin will auto-fluoresce as they pass through E. coli, Salmonella,
Millipore Milliflex Rapid
24-28 hr
60 per day
Filterable samples
1 CFU after growth
Millipore Milliflex Quantum
24-28 hr
60 per day
Filterable samples
1 CFU after growth
Pall Corporation GeneDisc Rapid Microbiology System
qPCR Estimation of cell count
3 to 8 hours including sample filtration, nucleic acid prep, and PCR 96 per PCR run
1 CFU after enrichment for 6 to 24 hours. >100 CFU without enrichment.
Instantaneous
Continuous monitoring
3.6 L/min
1 cell
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the laser. Those fluorescing biological particles are then counted in one of two size channels. The system can be integrated into an existing Environmental Monitoring data management platform or can be used with a local PC with available interface software. The sample material is collected on metal foil using impaction or filtration methods. Viability staining and Viable Staining and Imaging LED Raman Spectroscopy Identification and enumeration Cells from colony, liquid 3-10 min 300-600 ID's per hour medium, product/raw material, surfaces automated image analysis using dark field illumination detects viable particle quantity, shape, and size ranging from 0.5 m and larger. Raman spectroscopy is then performed on each viable particle and a spectral signature is provided. The spectral signatures are statistically correlated to a library of known microorganisms. Non-destructive for further analysis. Samples are prepared as per the compendial method and loaded into the system which manages the incubation and colony enumeration for 400 samples for 3-day EM Growth-based; Rapid Micro Biosystems The Growth Direct System automated detection of cellular auto-florescence Water analysis, Bioburden, Sterility, Environmental Monitoring Final results in one-half the time of the compendial method Positive results within hours Test 280 samples for 5-day water or bioburden tests 20-40 samples per day for Sterility Test Filterable samples Standard Environmental Monitoring samples the sample. Proprietary digital imaging technology automatically enumerates micro-colonies in one-half the time than traditional visual plate counting methods. The sample is collected onto a filter placed onto an agar medium cassette with an optically clear lid. Illumination with blue light excites micro-colonies to auto-fluoresce (without the addition of any reagents), which are enumerated by a CCD imaging system. The system automatically incubates and analyzes each cassette for the formation of micro-colonies over time. Particles that do not grow in size over time are ignored. Non-destructive; can continue to incubate media to obtain colonies for microbial identification. Air is drawn into the instrument and both viable and total particulates are simultaneously detected, sized and enumerated via a 685 nm laser diode (for particle sizing) and a 405 nm laser TSI Inc. BioTrak Real-Time Viable Particle Counter Light scattering Active air monitoring Instantaneous Continuous or episodic monitoring 28.3 L/min 1 cell Bacteria, yeast, mold, spores diode (for viability detection). The size range for detection is from 0.5 to 25 microns. The counting efficiency is 50% at 0.5 microns and 100% for particles > 0.75 microns (per ISO 21501-4 and JIS B9921). The system can be calibrated using NIST traceable standards. Also included is an integrated particle collection filter (gelatin filter) to capture microorganisms for subsequent growth and identification. Samples and reagent are added to a vial and mixed (5-10 min). The RAPID-B system analyzes the sample using flow cytometry, with results available within 3-5 minutes. Light scatter resolution of around 130nm shows separation of bacteria populations via size and refractive index alone. The system utilizes a multi-parametric detection approach: a phenotypic library to identify the bacteria, an immunoprobe to specifically tag the target organism and a DNA dye to ascertain the live or dead cells of the target organism. The workflow is also based on the limit of
rap.ID Particle Systems rap.ID
1 cell is identified and quantified
> 150 bacterial and spore entries; customizable
1 CFU
Bacteria, yeast, mold, all organisms that grow on agar media
Vivione Biosciences, LLC RAPID-B
Flow cytometry Incoming inspection, sterility confirmation, bioburden, drug development, process control
20 min for quantitative results Up to 8 hr for qualitative results
TB, Chlamydia and Staphylococcus for clinical samples. 96-160 per 8 hrs 75-225 uL 1 CFU after enrichment Salmonella, E. Coli O157, non-O157 STECs, Staphylococcus, and Vibrio for food samples
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detection (LOD) required and the target of interest (may include enrichment, filtration, centrifugation and dilution). k
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RMM Product Matrix - Rapid Microbiology and Rapid Microbiological Methods

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RMM Product Matrix - Rapid Microbiology and Rapid Microbiological Methods

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RMM Product Matrix - Rapid Microbiology and Rapid Microbiologic...

RMMS FOR MICROBIAL IDENTIFICATION

Sample Size or Type

40-160 uL of nucleic acid sample

10 copies per gene

Applied Biosystems MicroSEQ

PCR and gene sequencing Identification

80 per day. Higher with greater capacity capillary analyzers.

Raman spectroscopy Identification and enumeration

BD Diagnostic Systems Phoenix

Growth-based; biochemical utilization and antibiotic

100 per day

Cells from colony